Project Summary Tetherin is an interferon-inducible, antiviral protein with an extraordinarily broad mechanism of action. This cell- surface protein anchors budding viral particles to the cell surface as they emerge from the plasma membrane, preventing release from the cell. `Tethered? virions are endocytosed and destroyed, and tetherin also initiates a signal transduction cascade that triggers expression of additional innate immune defenses. Thus, tetherin is both a sensor and an effector, with the potential to inhibit all enveloped viruses that acquire their membranes by budding from the plasma membrane. This begs the question: how does the majority of enveloped viruses adapt to, and overcome, tetherin-mediated restriction? Tetherin's mechanism does not entail a specific interaction with a viral protein or gene product, making it difficult or impossible for viruses to escape directly by mutation and selection. A review of the now abundant literature reveals that most tetherin studies have focused on a few viruses encoding accessory proteins with known anti-tetherin functions. These include HIV and SIV, which use their Vpu and Nef accessory proteins, respectively, to reduce cell-surface tetherin levels. In contrast, the vast majority of retroviruses do not encode known or obvious accessory proteins with which to counteract host tetherin, including a majority of retroviruses in the alpharetrovirus, betaretrovirus and gammaretrovirus genera. For this R21 proposal, we plan to tackle this enormous knowledge gap by focusing first on viruses of the promiscuous RDR Interference Supergroup. The RDR supergroup comprises an extraordinary collection of viruses that includes gammaretroviruses, betaretroviruses, and several endogenous retroviruses (including the human Syncytin-1 gene, which originated as an endogenous retrovirus). RDR viruses colonize hosts as diverse as primates, carnivores, and birds. This proposal takes advantage of the fact that the RDR viruses share a genetically and functionally homologous env gene, and consequently, all RDR viruses tested thus far use the same receptor for viral entry (ASCT2). We discovered that Mason-Pfizer monkey virus (MPMV), a prototypical RDR virus, uses its Env glycoprotein to antagonize the tetherin homolog of its natural host, the rhesus macaque, providing a paradigm for understanding how other simple retroviruses may overcome tetherin in their respective hosts. We hypothesize that the anti-tetherin function of MPMV Env is common among RDR retroviruses (Aim 1). By leveraging the homology and functional similarities among RDR Envs, we will map the interface between Env and tetherin (Aim 2). Finally, we will test and confirm the mechanism(s) by which Env rescues virions from tetherin- mediated restriction (Aim 3).